Biomimetic mineralization of collagen fibrils via carboxymethyl chitosan stabilized liquid precursor

Abstract

Objective To test the hypothesis that the carboxymethyl chitosan (CMC) stabilized liquid precursors can induce biomimetic mineralization of collagen fibrils. Methods CMC-calcium phosphate solution turbidity assessment was used to determine the working concentration of CMC. Reconstituted 2-D typeⅠ collagen model and 3-D collagen membranes were treated with saturated calcium phosphate solution containing the above working concentration of CMC. Samples treated with traditional mineralization liquids (without CMC) were acted as control. The ultrastructural changes of the 2-D collagen were observed by transmission electron microscopy (TEM) and the mineral phase was determined by selected area electron diffraction (SAED) /energy dispersive X-ray analysis (EDXA) . The mineralization composition and degree of 3-D collagen film were analyzed by thermogravimetric (TG) and X-ray diffraction (XRD) . Results According to turbidity assessment of CMC, 200 μg/ml CMC was able to stabilizing calcium phosphate liquid for 7 days and selected for further experiments. Through TEM observation and SAED analysis, needle-like crystals along the long axis of the fibrils were observed in the intrafibrillar spaces of collagen fibrils on the 3rd day. SAED analysis showed the typical diffraction ring of hydroxyapatite. EDXA revealed that the Ca/P of minerals in the collagen fibrils was 1.56. As for 3-D collagen model, the mineral content of collagen membrane treated with the biomimetic mineralization liquid for 14 days was 18.39% via TG analysis and the mineral phase was confirmed to be hydroxyapatite according to XRD curves. Conclusions Carboxymethyl chitosan can induce the biomimetic mineralization of collagen fibrils through stabilizing of liquid precursor and synthesize biomimetic collagen-hydroxyapatite complex with relatively higher degree of mineralization. Key words: Carboxymethyl chitosan; Collagen; Biomimetic mineralization; Polymer-induced liquid precursor